Tissue transplantation is a rapidly growing therapeutic field as a result of improvements in surgical procedures, immuno-suppressive treatments, and increased knowledge of the graft-host interaction. Despite major advances, problems associated with tissue transplantation include inflammation, degradation, calcification, and rejection of the transplanted tissue.
There are several applications for tissue transplantation. Heart malfunction due to heart valve disorders can often be treated by surgically implanting a prosthetic valve. Treated tissue derived from porcine aortic valves or bovine pericardium, is currently used in prosthetic heart valves. Tissue must be stabilized prior to implantation into an animal different from the donor animal. This process of stabilization is known in the art as fixation. The fixation process makes the tissue more resistant to degradation upon implantation. It also makes it non immunogenic so that it will not be rejected by the host human body.
Generally, the fixation process operates by blocking reactive molecules on the surface of and within the donor tissue, thereby rendering it substantially non-antigenic and suitable for implantation as well as crosslinking the collagenous matrix providing stability. Collagenous tissue, usually the major component of a typical bioprosthesis, can be fixated by a number of methods, including treating the material with aldehydes. Glutaraldehyde is the most common reagent used in the fixation of animal tissue. Glutaraldehyde is easily available, bifunctional, inexpensive and reacts under physiological conditions with primary amine groups of collagen molecule. However, there are some significant problems with glutaraldehyde fixation method. The Schiff-base bond between collagen and glutaraldehyde is unstable in nature. Glutaraldehyde polymerizes in water to produce a water soluble polyether polymer. The glutaraldehyde treated tissue is more susceptible to calcification. Glutaraldehyde is cytotoxic. Finally, the polymeric product of glutaraldehyde produces glutaraldehyde by depolymerization reaction of its polymeric form, the polyether. The leaching of cytotoxic glutaraldehyde is believed to prevent cellular growth on the bioprosthesis necessary for long term biocompatibility.
The problems associated with the glutaraldehyde fixed tissue led to the development of alternative tissue fixatives. The state of the art of tissue fixation is well documented in the literature (recent review article by Eugene Khor, Tissues, Volume 18, page 95-105 and reference therein). Other bifunctional or polyfunctional tissue reactive reagents known in the art include polyepoxides, diisocyanates, polyfunctional acids, a di-functional acid, and 1,6-hexane diamine, carbodiimides and photooxidation using organic dyes. These treatments have varying success in replacing glutaraldehyde as a tissue crosslinking agent, but none of these has achieved the success of the glutaraldehyde fixation.
Efforts at retarding the calcification of bioprosthetic tissue have been numerous in recent years. The techniques resulting from these efforts may be broadly divided into two categories; those involving the pre- or post-treatment of glutaraldehyde-fixed tissue with one or more compounds that inhibit calcification (or modify the fixed tissue to be less prone to calcification) and those involving the fixation of the tissue with compounds other than glutaraldehyde, thereby reducing calcification.
The former category of techniques includes, but is not limited to, treatment with such compounds as:
a) detergent or surfactant, after glutaraldehyde fixation; PA1 b) diphosphonates, covalently bound to the glutaraldehyde-fixed tissue or administered via injection to the recipient of the bioprosthesis or site-specifically delivered via an osmotic pump or controlled-release matrix; PA1 c) amino-substituted aliphatic functional acid, covalently bound after glutaraldehyde-fixation; PA1 d) sulfated polysaccharides, especially chondroitin sulfate, after glutaraldehyde fixation and preferably followed by treatment with other matrix-stabilizing materials; PA1 e) ferric or stannic salts, either before or after glutaraldehyde fixation; PA1 f) polymers, especially elastomeric polymers, incorporated into the glutaraldehyde-fixed tissue; or PA1 g) water-soluble solutions of a phosphate ester or a quaternary ammonium salt or a sulfated higher aliphatic alcohol, after glutaraldehyde-fixation. PA1 a) treatment by soaking the bioprostetic tissue in an aqueous solution of high osmolality containing a photo-oxidative catalyst and then exposing said tissue to light thereby fixing the tissue via-photo-oxidization; and PA1 b) fixation via treatment with a polyepoxy compound, such as polyglycidyl ether (polyepoxy) compound.
The latter category of techniques for reducing the calcification or bioprosthetic tissue, i.e., techniques involving the fixation of the tissue with compounds other than glutaraldehyde, includes but is not limited to, the following:
A recently reported fixation method uses a coupler and a coupling enhancer with or without one or more coupling agents. It fixes the tissue by linking the amine and the carboxyl moieties through amide bonds either directly, or indirectly when coupling agents form bridges. Tissue is fixed using the coupling agents such as 1,6-hexane diamine and suberic acid in the presence of the coupler such as 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide hydrochloride and a coupling enhancer N-hydroxysulfosuccinimide.
In most cases, investigations related to glutaraldehyde-associated sympotomatology have been limited to specific problems such as calcification and have not addressed the entire spectrum of symptoms. Thus, while the problem of calcification of glutaraldehyde-fixed bioprostheses has received a great deal of attention, the proposed solutions have generally failed to address any other complications presented by the presence of glutaraldehyde, such as toxicity, immune reactions and degeneration. Glutaraldehyde released from the tissue is cytotoxic and prevents the formation of endothelial cell growth on the bioprosthesis necessary for long-term durability. This persistent damage to the implant and surrounding tissue due to the long-term slow release of glutaraldehyde may be fully eradicated only by using a fixation process that does not include glutaraldehyde. The complexity and gravity of the clinical problems resulting from glutaraldehyde-preserved bioprostheses warrant the search for an alternative fixation method.
What is needed in the art is a fixation method that can 1) be performed quickly and if necessary in vitro, 2) does not introduce cytotoxic chemicals or other material that will hinder the adhesion and growth of host endothelial cells, 3) that provides adequate fixation under conditions of normal pH, and 4) does not promote calcification.